The RNA interference (RNAi) pathway is an evolutionarily conserved mode of gene regulation. The RNAi process is initiated by double-stranded RNA (dsRNA) produced from various exogenous or endogenous sources (e.g., experimental introduction, viral infection). The dsRNA is cleaved by Dicer to generate 20-25 nucleotide small-interfering RNA (siRNA) duplexes. These duplexes are then loaded onto the RNA-induced silencing complex (RISC), and before RISC is activated, the passenger/sense strand of the duplex is removed. The single guide/antisense strand remains associated with RISC and directs cleavage of the target mRNA. Thus, duplexed siRNA have become an important tool for both research and nucleic acid-based therapeutics.
RNAi gene silencing can occur via single-stranded or double-stranded RNA molecules. In the last ten years, it has been reported that single-stranded antisense siRNA are almost as potent as the siRNA duplex (see, e.g., Schwarz et al., 2002, Mol. Cell. 10:537-548; Martinez et al., 2002, Cell 110:563-574; Amarzguioui et al., 2003, Nucleic Acids Res. 31:589-595; and Holen et al., 2003, Nucleic Acids Res. 31:2401-2407). There are benefits in utilizing single-stranded RNA molecules, as opposed to duplexed versions, for gene silencing. Their lower molecule weight may make them easier to cross cell membranes. Single-stranded RNA molecules are also half the mass and volume of duplexed siRNA, implicating a manufacturing cost advantage. Thus, there remains a heightened interest in formulating new and advantageous design features suitable for single-stranded RNAi molecules.